The longterm objective of our work is to understand the role of Ca metabolism in cardiac failure. Such an understanding is critical to the design of effective interventions for its treatment. Our strategy is to investigate the role of Ca metabolism in failure at the cellular level, using isolated ventricular cells. We have developed a procedure for isolating Catolerant cells from adult rat hearts, and from experiments conducted over the past six years we have established that the cells have metabolic and functional properties similar to those of cells in the whole heart. We have pursued the development of methods for evaluating the function of isolated heart cells. We have begun to investigate the Na and Ca metabolism of the cells more fully, along with their contractile function, as measured by laser light diffraction. Cellular Ca uptake is measured 45Ca and by the intracellular Ca indicators quin2 and indol. We have also developed an assay for slow Ca channel activity, using 54Mn. The continuation of this work constitutes the first broad objective of this proposal. We have recently found that ATP depletion strongly inhibits Ca influx into cells, much of it Na/CA exchange. The mechanism of this must be clarified. We also find that rapid perfusion of cells with a caffeine medium induces, after a delay, a contracture which can be as rapid as that induced by stimulation. The mechanism of this trigger must be clarified. The caffeine contracture has potential as an assay for the Ca pool in the sarcoplasmic reticulum and will be used to test the theory that beatdependent changes in contractility reflect the size of this pool. We will continue to pursue methods development, by measuring membrane potentials in the cells with the tetraphenylphosphanium ion, and by measuring intracellular Ca transients with quin2. These, along with the methods we already have, will give us a comprehensive arsenal for the evaluation of Ca metabolism in isolated cells, not only from animals but also from humans. We have begun to isolate cells from failing human hearts. We have had some success, and prospects are good that a viable preparation is achievable. Our second broad objective is to optimize this isolation procedure and then to characterize the contractile function and Ca metabolism of human cells from normal and cardiomyopthic hearts. Thus, our two objective mesh: the human cells will be studied in the context of the animal cells, using the methodologies and concepts worked out for the latter.